Tubular insulin-induced gene 1 deficiency promotes NAD+ consumption and exacerbates kidney fibrosis

Profibrotic proximal tubules (PT) were identified as a unique phenotype of proximal tubule cells (PTCs) in renal fibrosis by single-cell RNA sequencing (scRNA-seq). Controlling the process of renal fibrosis requires understanding how to manage the S1 subset’s branch to the S3 subset rather than to the profibrotic PT subset. Insulin-induced gene 1 (Insig1) is one of the branch-dependent genes involved in controlling this process, although its role in renal fibrosis is unknown. Here, we discovered that tubular Insig1 deficiency, rather than fibroblast Insig1 deficiency, plays a detrimental role in the pathogenesis of renal fibrosis in vivo and in vitro. Overexpression of Insig1 profoundly inhibited renal fibrosis. Mechanistically, Insig1 deletion in PTCs boosted SREBP1 nuclear localization, increasing Aldh1a1 transcriptional activity, causing excessive NAD+ consumption and ER enlargement, as well as accelerating renal fibrosis. We also identified nicardipine as a selective inhibitor of Aldh1a1, which could restore NAD+ and maintain ER homeostasis, as well as improve renal fibrosis. Together, our findings support tubular Insig1 as a new therapeutic target for chronic kidney disease (CKD).

5th Jan 2024 1st Editorial Decision 4th Jan 2024 Decision on your manuscript EMM-2023-19208 Dear Dr. Yu, Thank you for submitting your manuscript "Tubular insulin-induced gene 1 deficiency promotes NAD+ consumption and exacerbates kidney fibrosis" to EMBO Molecular Medicine.I have now carefully read your manuscript and discussed it with my colleagues.I regret to say that we all agree that the manuscript is not well suited for publication in EMBO Molecular Medicine and therefore have decided not to proceed with peer review.
In this study, you identify Insig1 as an ER stress related gene that is downregulated in patients with chronic kidney disease and you show that loss of Insig1 in proximal tubule cells in mice promotes renal fibrosis upon UUO.We appreciate that you report that mechanistically Insig1 expression is regulated by Aldh1a1, and that you identify a new Aldh1a1 inhibitor, nicardipine, that reduces fibrosis in mice and the expression of fibrotic genes in cells.However, we feel that the conceptual advance and clinical significance presented in the work remains limited given that there are several other Aldh1a1 inhibitors and that a therapeutic improvement as a result of nicardipine administration over e.g.NCT-501 has not been demonstrated.Thus, we are unfortunately not persuaded that your manuscript provides the sort of clinical impact and advance we would expect in an EMBO Molecular Medicine article.I am sorry that I could not bring better news this time but hope that this negative decision does not prevent you from considering our journal for the publication of your future studies.

Yours sincerely, Poonam Bheda, PhD Scientific Editor EMBO Molecular Medicine
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Dear Poonam Bheda, Thank you for reviewing my manuscript.I regret that my research has not been able to get your approval.However, I would like to explain the questions you raised about my research.
Firstly, we discovered that Insig1 deletion in PTCs boosted SREBP1 nuclear localization, increased Aldh1a1 transcriptional activity, caused excessive NAD+ consumption and ER enlargement, and accelerated renal fibrosis, not "Insig1 expression is regulated by Aldh1a1".
Secondly, the equilibrium dissociation constant (KD) value of Aldh1a1 with nicardipine was 1.98E-06 M, indicating strong binding between nicardipine and Aldh1a1, while the KD value of Aldh1a1 with NCT501 was 3.31E-05 M. What's more, Nicardipine is effective in the treatment of blood pressure in clinical, but here, we found that it effectively attenuated CKD, expanding its clinical indications to treatment of CKD patients with hypertension, which demonstrated the clinical significance in the work.Futhermore, the therapeutic improvement of nicardipine administration over e.g.NCT-501 had been demonstrated pathological staining (Specific statistics were not provided).Later, we can compare the therapeutic improvement of two drugs in a series of experiments.
We still sincerely hope that this work will attract your interest.We appreciate you considering it for publication in the EMBO Molecular Medicine journal, and we hope to hear from you soon.Thank you for the submission of your manuscript to EMBO Molecular Medicine.We have now received feedback from the three reviewers who agreed to evaluate your manuscript.As you will see from the reports below, the referees acknowledge the interest of the study and are overall supportive of your work; however they also comment on multiple aspects of the manuscript that should be strengthened in a revision.
In particular, improvements in the presentation and figures are necessary.In addition, the revisions should include a comparison of nicardipine with NCT501 (Reviewer 1), analyze your transcriptomics data for differential expression of Insig1 (Reviewer 2), and investigate the effect of Insig PTC-cKO on E-cadherin and vimentin expression (Reviewer 3).In a cross-consultation session between the editor and reviewers, there was discussion as to whether renal glomeruli need to be included in the Sirius Red staining images as the study primarily focuses on observing renal interstitial fibrosis induced by UUO and partial nephrectomy, rather than glomerular damage.Therefore for this concern, you may include experimental evidence or include a justification to Reviewer 2 as to why this may not be necessary.
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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection.Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status.Li et al. carried out a comprehensive analysis of Insig1 and Aldh1a1 in kidney disease.In the kidney disease field, there is still an open question about which genes are in charge of renal fibrosis and how they realize that.In this study, their result demonstrated that Insig1 deficiency enhanced the nuclear localization SREBP1 and the transcriptional activity of its target gene Aldh1a1, causing excessive NAD+ consumption, ER stress, and ultimate renal fibrosis.Overall, the results support their conclusions.In particular, the function of Aldh1a1 in regulating NAD+ and further renal fibrosis provides important novelty.Due to some minor issues, I suggest giving a chance for minor revision.If these minor issues are solved, the manuscript will be acceptable for publication.
Minor issues: 1.The introduction section is too long, should be cut by approx.10-20%.2. Regarding human biopsies, 90% of samples were from children under 18 years, statements in the manuscript, such as "CKD patients" should be changed to "Children with kidney disease".3. The method to isolate proximal tubules and fibroblasts in the kidney are not described.4. Figure S5: for those not knowing lipid metabolism, please explain the chosen analysis to address whether Insig1 effect is dependent on lipogenic activity.5. Please explain how to choose Aldh1a1 as a downstream target and exclude others?6.What are the benefits of Nicardipine over NCT501 as a new Aldh1a1 inhibitor?7. English writing needs improvement.

Referee #2 (Remarks for Author):
There are obvious errors or lack of rigor in the paper graphics.1.In Figure 1m, the IHC image should be white balanced to ensure consistent background brightness.2.In Figure 2d, in the FN detection of Insig1△Kap mice with sham operation, in order to be focused on renal medulla, there should be focused on the field of view with glomeruli.In Figure 2f, the bands in western blot show that the expression of GAPDH in the groups of the sham and UUO is uneven.It is recommended to replaced.3.In Figure 3a, b&c, the group of 5/6nX treated Insig1△Kap mice, there might be a mouse which should be removed, because the renal function in each image was only one which was dispersed from other five ones.In Figure 3c, in the Sirius red detectionof Insig1flox/flox and Insig1△Kap mice treated with 5/6Nx, in order to be focused on renal medulla, there should be focused on the field of view with glomeruli.4.In Figure 4b, the bands in western blot show that the expression of GAPDH in the groups of the sham and UUO is uneven.5.In Figure 5d, the representative images of FN staining in si-NC stimulated by TGF-β1, the number of cells is significantly less than other groups and needs to be adjusted to be consistent.In Figure 5i, the number of cells in both of the NC and Insig 1 overexpressed groups were significantly less than other groups.It seems like that there was cytotoxicity of the vehicle.6.In Figure 6g, there should be corresponding statistical charts showing the obvious expression levels between the si-NC and si-insig1.The entire text should be drawn as a mechanism diagram rather than being divided into several parts, such as Figure 6i and Figure 8f.7.In Figure 7a, the bands in western blot show that the expression of GAPDH in the groups were uneven.In Figure 7k, the number of cells in both of the NC and Aldh1a1 overexpressed groups were significantly less than other groups.It seems like that there was cytotoxicity of the vehicle.
expression, or TG content were not further increased by Insig1 deletion in PTCs.In line with this finding, when Insig1 was knocked down in TKPTS cells, TOFA (an inhibitor of acetyl-CoA carboxylase-α) and C75 (an inhibitor of fatty-acid) were unable to block the profibrotic effect of TGF-β1.Thus, the profibrotic effect of Insig1 deletion in PTCs did not appear to be linked to lipogenic activity.Response: Thanks for your valuable comments and suggestion.We have deleted it in the revised MS.
(1) NCT501 is a potent and selective inhibitor of Aldh1a1, its half-life is still very short (T 1/2 < 1 h) and clearance level was very high (CL = 98 mL/min/kg), which suggests that NCT501 is well absorbed and distributed but rapidly metabolized and/or excreted.Therefore, further work focused on the improvement of the half-life and oral bioavailability is beneficial and worthy of done (Li et al, 2021).
(2) Nicardipine, a drug commonly used to treat hypertension in clinical, was found to be a specific inhibitor of Aldh1a1.Furthermore, it binds to Aldh1a1 with greater stability than NCT501.
(3) To evaluate the therapeutic improvement of nicardipine administration over NCT501, we compare the therapeutic effect of two drugs in the UUO-induced 2) In Figure 2d, in the FN detection of Insig1△Kap mice with sham operation, in order to be focused on renal medulla, there should be focused on the field of view with glomeruli.In Figure 2f, the bands in western blot show that the expression of GAPDH in the groups of the sham and UUO is uneven.It is recommended to replaced.Following your comments, we re-examined the samples using WB method and changed all the bands in Fig 2F .3) In Figure 3a, b&c, the group of 5/6nX treated Insig1△Kap mice, there might be a mouse which should be removed, because the renal function in each image was only one which was dispersed from other five ones.In Figure 3c, in the Sirius red detection of Insig1flox/flox and Insig1△Kap mice treated with 5/6Nx, in order to be focused on renal medulla, there should be focused on the field of view with glomeruli.Response: Thanks for your valuable comments.We have carried out the experiment again and changed it in the revised MS. 5) In Figure 5d, the representative images of FN staining in si-NC stimulated by TGF-β1, the number of cells is significantly less than other groups and needs to be adjusted to be consistent.In Figure 5i, the number of cells in both of the NC and Insig 1 overexpressed groups were significantly less than other groups.It seems like that there was cytotoxicity of the vehicle.9) In Supplementary Figure S7a, the images in Masson staining should be white balanced to ensure consistent background brightness.In Supplementary Figure S7e and S11c, the images in three groups were not at the same magnification factor.

Response
Response: Thanks for your valuable comments and suggestion.Following the comment of reviewer #1, we compare the therapeutic effects of two drugs in the UUO-induced CKD model in order to evaluate the therapeutic improvement of nicardipine administration over NCT501 (Fig EV5H and I).
Thus, we removed the Masson staining images of NCT501 in Figure S7a.
Furthermore, the same rulers were automatically inserted by ZEN program, which surprised us even more with the data from Supplementary Figure S7e and S11c.We carried out the experiment again and re-selected the representative images (Fig EV5C and G).10) In Supplementary Figure S9a, the bands of ip:BIP were unclear and fuzzy.
In Supplementary Figure S10D, the bands in western blot show that the expression of GAPDH in the groups were uneven.
Response: Thanks for your comments.In the revised MS, as shown in Fig 8J and Appendix Figure S2E, the prior results were replaced after we collected the cell samples again and performed the WB experiment again.

Responses to reviewer #3：
1) In Figure 2d  Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.We have now received the enclosed reports from the referees that were asked to re-assess it.As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments and appropriate response to reviewers: 1) Please check the "Author Checklist" carefully and complete all relevant questions.Currently the author name and manuscript number are missing.2) Abstract: Please check whether "Profibrotic proximal tubular (PT) were..." is correct.It seems like it should be "Profibrotic proximal tubules were...".Please also define PTCs in the abstract of the main text.
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-Human research participants: Please be sure to include a sentence in the Materials and Methods as to whether the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.-Please note that a separate 'Data Information' section is required in the legends of figures 2d-f, j; 3a-d, f; 5b-j; 6d-k; 7c-f, i; 9g-i, k, o-q; EV 2a, c; EV 3a, c-g.-Please note that in the legend of figures 1d-e, the red arrows are incorrectly mentioned as red asterisks.This needs to be rectified.
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-Please note that the box plots need to be defined in terms of minima, maxima, centre, bounds of box and whiskers, and percentile in the legends of figures 1h; 8b.
-Please note that information related to n is missing in the legends of figures 1l; 8b; EV 1f; EV 4a, e, h.-Please note that n=2 in figures 5a; 9b.Error bars cannot be calculated for measurements with n=2.
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-Please note that the red arrows are not defined in the legends of figures 2h; 3g; 7g; 9j.This needs to be rectified.
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-Synopsis text: We would suggest rephrasing "worsened NAD+ consumption" as it is unclear whether this is increased or decreased.Our suggestion is the following: "Chronic kidney disease (CKD) mice with loss of Insulin-induced gene 1 (Insig1) in proximal tubule cells exhibit increased NAD+ consumption and worsened renal fibrosis, indicating that activation of Insig1 may offer a new approach to treating CKD." -Please check your synopsis text and image before submission with your revised manuscript.Please be aware that in the proof stage minor corrections only are allowed (e.g., typos).10) As part of the EMBO Publications transparent editorial process initiative (see our policy here: https://www.embopress.org/transparent-process#Review_Process),EMBO Molecular Medicine will publish online a Peer Review File (PRF) to accompany accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.Let us know whether you agree with the publication of the PRF and as here, if you want to remove or not any figures from it prior to publication.Please note that the Authors checklist will be published at the end of the PRF.11) Please provide a point-by-point letter INCLUDING my comments as well as the reviewer's reports and your detailed responses (as Word file).This study maintained the homeostasis of tubular Insig1 is a novel mechanism in mediating CKD, but also identify nicardipine as a specific Aldh1a1 antagonist for treating CKD.

Referee #2 (Remarks for Author):
There were still obvious errors in the paper graphics： 1.In Figure 1m, the IHC image in health group, the renal tubules show significant swelling and occlusion, which is not the normal morphology of the renal tubules.2.In Figure 2g, hollow and solid points, green and yellow bar charts represent different groups and should be distinguished from the Figure 2e.A hollow point should not represent Sham group and Insig 1 flox/flox+UUO group at the same time in Figure 2. In the following figures, there was the same questions.I suggest mecessary modifications should be made.
Referee #3 (Comments on Novelty/Model System for Author): No further comments.

Responses to reviewer #1：
I have no further comment, thank you.

Response:
We appreciate the valuable comments to improve the quality this manuscript and the kind support on this research work.

Responses to reviewer #2：
1.In Figure 1n, the IHC image in health group, the renal tubules show significant swelling and occlusion, which is not the normal morphology of the renal tubules.
Response: Thanks for your valuable comments and suggestion.We have carefully examined the IHC images of the normal group, and found that some renal tubules were morphologically occlused and swollen.This was mainly because normal kidney tissue was taken from paracarcinoma.We have replaced the representative image in

Responses to reviewer #3：
No further comments.

Response:
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10) We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be cited as 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.
5) Please explain how to choose Aldh1a1 as a downstream target and exclude others?Response: Thanks for your valuable comments and suggestion.We have added it in the Fig 6C and D. 6) What are the benefits of Nicardipine over NCT501 as a new Aldh1a1 inhibitor?
CKD model.As shown in Fig EV5H-I, compared to NCT501, nicardipine treatment significantly reduced collagen deposition and the fibrotic indicators expression in the kidneys following UUO surgery.7) English writing needs improvement.Response: Thanks for the comment.In this revised MS, we thoroughly checked and corrected the errors in typing and grammar.Reference Li B, Yang K, Liang D, Jiang C, Ma Z (2021) Discovery and development of selective aldehyde dehydrogenase 1A1 (ALDH1A1) inhibitors.European journal of medicinal chemistry 209: 112940 Responses to reviewer #2： 1) In Figure 1m, the IHC image should be white balanced to ensure consistent background brightness.Response: Thanks for your suggestion.Following your suggestion, in the revised MS, we have repeated the immunohistochemical staining, using a pathological scanner to scan the slices for imaging, and changed the previous images in Fig 1N.

:
Thanks for your valuable comments.In Fig 2D, we have selected a more representative image in the revised MS.Even though there were the same amount of loaded proteins in each lane in Fig 2F, the expression of GAPDH is unequal due to the consideration of additional external factors.

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Thanks for your valuable comments.Following your suggestion, a mouse with renal function was distributed among the other five has been removed in Fig 3. Additionally, in the revised MS, we have carried out the experiment again and re-selected the representative images in Fig 3C.
4) In Figure4b, the bands in western blot show that the expression of GAPDH in the groups of the sham and UUO is uneven.

Response:
Thanks for your valuable comments and suggestion.In the revised MS, we have re-selected the representative images in Fig 5D and I. 6) In Figure 6g, there should be corresponding statistical charts showing the obvious expression levels between the si-NC and si-insig1.The entire text should be drawn as a mechanism diagram rather than being divided into several parts, such as Figure 6i and Figure 8f.Response: Thanks for your suggestion.As shown in Fig 6I, we have added the statistical charts.We acknowledge your point of view and have removed the small diagrams in the revised MS.We divided them up to help the reader understand the results.7) In Figure 7a, the bands in western blot show that the expression of GAPDH in the groups were uneven.In Figure 7k, the number of cells in both of the NC and Aldh1a1 overexpressed groups were significantly less than other groups.It seems like that there was cytotoxicity of the vehicle.Response: Thanks for your important comments.In the revised MS, we have carried out the experiment again and changed the bands and representative images in Fig 7A and J. 8) Regarding the differential expression of Insig1, is there any indication in the transcriptomic data of the UUO model.Response: Thanks for your important comments.Following your comments, we used an online database (GSE145053) to confirm the decrease in Insig1 mRNA levels in UUO models (Fig 1M).
, the Sirius Red staining of the insig1 flox/flox UUO group shows too little collagen deposition (bright red area).Typically, successful UUO results in severe fibrotic lesion.As demonstrated in Figure 7c, the Sirius Red staining and FN staining of the WT-UUO group showed a typical kidney fibrosis.Please select a more representative image.Response: Thanks for your valuable comments and suggestion.In the revised MS, we have re-selected the representative images in Fig 2D.
2) In Figure2f, the protein bands of GAPDH are visibly inconsistent, suggesting potential issues with the accuracy of protein loading for gel electrophoresis.Please clarify.Response: Thanks for your valuable comments and suggestion.Even though there were the same amount of loaded proteins in each lane in Fig 2F, the expression of GAPDH is unequal due to the consideration of additional external factors.Following your comments, we re-examined the samples using WB method and changed all the bands.3) It is well established that the tubule epithelial-to-mesenchymal transition (EMT) of plays a crucial role in the pathogenesis of renal fibrosis.Therefore, it would be better if the authors could investigate the effects of insig1 PTC-cKO on the expression of E-cadherin and vimentin in kidneys.Response: Thanks for your valuable comments.Following your comments, we have investigated the effects of Insig1 PTC-cKO on the expression of E-cadherin and vimentin in the kidneys of Insig1 flox/flox and Insig1 Kap mice after UUO or FA treatment.Following UUO, the kidneys of Insig1 Kap animals displayed considerably lower expression of E-CADHERIN and significantly higher expression of VIMENTIN (Vim) (Fig 2E and F).Furthermore, after FA treatment, Insig1 deficiency in PTCs increased expression of Vimentin ( 6) Please place individual sections of the manuscript in the following order: Title page -Abstract & Keywords -Introduction -Results -Discussion -Materials & Methods -Data Availability -Acknowledgements -Disclosure and Competing Interests Statement -The Paper Explained -For More Information -References -Figure Legends -Expanded View Figure Legends.7) For the figures and figure legends, please take care of the following:

I
look forward to reading a new revised version of your manuscript as soon as possible.Instructions to submit your revised manuscript *** ***** Reviewer's comments ***** Referee #1 (Remarks for Author): I have no further comment, thank you.Referee #2 (Comments on Novelty/Model System for Author): Fig 1N.
Figure 2g, hollow and solid points, green and yellow bar charts represent different groups and should be distinguished from the Figure 2e.A hollow point should not represent Sham group and Insig 1 flox/flox+UUO group at the same time in Figure 2. In the following figures, there was the same questions.I suggest mecessary modifications should be made.Response: Thanks for your valuable comments and suggestion.According to your suggestion, we have updated Fig 2G, Fig 3E, Fig 4F-H, Fig 6K, Fig 7F,K,M, Fig 8C,E, Fig EV2D-F, Fig EV3E,F, Fig EV4H.
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